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Electronic Supplementary Information (ESI) for

Multiplexed Femtomolar Quantitation of Human Cytokines in a

Fluoropolymer Microcapillary Film

Ana P. Castanheira,a Ana I. Barbosa,b Alexander D. Edwardsac and Nuno M. Reis*ab

a Capillary Film Technology Ltd, West Sussex RH14 9SJ, United Kingdom b Department of Chemical Engineering, Loughborough University, Leicestershire LE11 3TU, United Kingdom. E-mail: n.m.reis@lboro.ac.uk; Tel: +44 (0)1509 222505; Fax: +44 (0)1509 223923 c Reading School of Pharmacy, University of Reading, Reading RG6 6AD, United Kingdom. E-mail: a.d.edwards@reading.ac.uk; Fax: +44 (0)118 931 4404; Tel: +44 (0)118 378 4253

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2015

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TABLE OF CONTENTS

SUPPLEMENTARY EXPERIMENTAL DESIGN

Multi-syringe aspirator (MSA) device

Fig. S1 Overview of Multi-Syringe Aspirator (MSA) device and miniaturised

fluoropolymer MCF platform.

Singleplex IL-1 Immunoassay Optimisation

Table S1 Optimised assay conditions for human IL-1 singleplex measurement

Fig. S2 Cytokine test strips imaged after OPD conversion, showing the relevance

of washing before addition of antigen. The strips were incubated with decreasing

concentration of antigens from the left to right hand side of the picture.

Selection of enzymatic amplification system

Fig. S3 Comparison of different Peroxide enzymes tested for colorimetric

amplification:

Assay Variability

Qualitative Duplex Assay

Quantitative Triplex Assay.

SUPPLEMENTARY RESULTS

Assay Optimisation

Fig. S4 Optimisation of IL-1 cytokine immunoassay measurement in the

fluoropolymer MCF.

Limit of Quantitation of the Colorimetric Detection Device

Fig. S5 Response curves for DAP detection in the MCF with flatbed scanner and

96 well MTP with microplate reader (450 nm).

Qualitative Duplex Detection

Fig. S6 Qualitative duplex assay for detection of IL-1β and IL-6 cytokines.

Optimisation of the Triplex Assay.

Fig. S7 Comparison of background in Triplex Cytokine Assay.

SUPPLEMENTARY REFERENCES

 

SUPPL

Multi-s

This wa

an array

fit seals

Fig. S1

MCF p

disposab

MicroC

Singlep

Several

detectio

flatbed s

volume

well, all

that cou

tested a

respect t

LEMENTAR

yringe aspi

as first prese

y of 8, 1 ml

and a custo

Overview

latform. a)

ble 1ml syr

apillary Film

plex IL-1 I

parameters

on and quan

scanner. Be

(SAV) rati

l immunoas

uld be detec

and shortlis

to maximum

RY EXPER

irator (MSA

ented by Ba

plastic syri

omised samp

of Multi-S

MSA devi

ringes and p

m (MCF). c

Immunoass

s were teste

ntification i

ecause of the

o and light

ssay conditi

cted using a

sted from a

m signal-to-

RIMENTA

A) device.

arbosa et al

inges that is

ple well, as

Syringe Asp

ice capable

pre-coated

c) Push-fit se

say Optimis

ed in order

in the MCF

e significant

path distan

ions had to

flatbed sca

an extensive

-noise ratio o

L DESIGN

l,.1 and cons

s interfaced

shown in F

pirator (MS

of doing 8

30 mm flu

eals holding

sation

to optimise

F based on

t difference

nce in small

be optimise

anner. Table

e initial sc

or signal-to

N

sists of a se

d with the pr

ig. S1.

A) device

80 simultan

uoropolymer

g 8 cytokine

e and impr

n colorimetr

s in the volu

capillaries

ed in order

e S1 summa

reening. Al

-background

emi-disposab

re-coated M

and miniatu

neous tests

r MCF strip

e test strips

rove the sen

ric ELISA

ume of reag

compared t

to produce

arises the fu

ll paramete

d ratio.

ble device c

MCF strips u

 

urised fluor

using an a

ps. b) Fluor

nsitivity of

quantitation

gents, surfac

to 96-micro

a colorimet

ull range for

er were opt

S3

containing

using push

ropolymer

array of 8

ropolymer

cytokines

n using a

ce-area-to-

otiter plate

tric signal

r variables

timised in

 

Th

length w

was incu

concent

1β capA

200 pg/

were co

was blo

Bovine

respect

concent

It

the goo

optimiza

the bloc

he optimum

with 0, 10, 2

ubated with

tration of 0,

Ab. On both

/ml. To test

oated with 1

ocked using

Serum (FB

to the initia

trations and

Table S1.

is importan

od perform

ation the tw

cking and th

m capAb con

20 and 40 μ

h PBS only.

5, 10 and 2

h experimen

the perform

10 μg/ml of

Bovine Se

BS) and 0.0

al rate of ab

blocking so

Optimised a

nt to mention

mance and

wo approach

he addition o

ncentration w

μg/ml of IL

On a separ

20 μg/ml usi

ntal sets, the

mance of dif

f IL-1β capA

erum Album

2% of Sodi

sorbance ge

olutions Ext

assay condit

n that a was

reduced v

hes were tes

of the antige

was determi

-1β mAb. F

rate experim

ing MCF str

e concentrat

fferent bloc

Ab. Followi

min 1% (BS

ium Azide

eneration. D

trAvidin wa

tions for hu

shing step b

variability

sted and it w

en was very

ined by coa

For the cont

mental set, d

rips coated w

tion of IL-1

cking solutio

ing 2 hours

SA) and the

(NaN3). Th

During the o

s used as en

uman IL-1

before the an

of the ass

was verified

important (

ating in dupl

trol (0 μg/m

detAb was te

with 10 μg/m

1β recombin

ons, two 12

s of incubat

second on

he results w

optimisation

nzyme.

singleplex m

ntigen incub

say. During

d that the w

(Figure S2).

licate 8 strip

ml of capAb

ested in dup

ml or 20 μg

nant protein

2 cm long M

tion, one of

e with 2.5%

were then an

n of capAb a

measuremen

bation was c

g prelimina

washing step

S4

ps of 3 cm

b) the strip

plicate at a

g/ml of IL-

n used was

MCF strips

f the strips

% of Fetal

nalysed in

and detAb

nt

crucial for

ary assay

p between

 

Fig. S2

before a

from the

Selectio

Peroxid

a range

length o

procedu

either H

(HSS-H

solution

HSS-HR

results o

signal in

Streptav

Sensitiv

NeutrAv

affinity

biotin/st

for strep

avidin (

of time.

Cytokine t

addition of

e left to righ

on of enzym

dase is a pop

of polymer

of MCF w

ure to the ca

High Sensiti

HRP). Each

n) was also

RP were tes

obtained wi

ntensity, ba

vidin-HRP r

vity NeutrAv

vidin each w

and selec

treptavidin

ptavidin (2.

7.5 × 10−8),

test strips im

antigen. Th

ht hand side

matic ampli

pular enzym

ized and co

was directly

apAb coatin

ivity NeutrA

enzyme w

prepared fo

sted for a c

th 4 μg/ml

ackground i

revealed in

vidin-HRP

were reporte

ctivity.2  Pir

and biotin/e

.4 × 10−6 s−

which can

maged after

he strips wer

of the pictu

fication sys

me in ELISA

njugated pe

coated wi

ng. The MC

Avidin–HR

was tested

or each enzy

concentratio

of ExtrAvid

ntensity and

ncreased sig

and ExtrAv

ed by the m

ran, et al2

egg avidin c−1) was abo

explain the

r OPD conv

re incubated

ure.

stem

A for presen

eroxidase en

ith 2 μg/m

CF was then

RP (HSN-HR

in duplicat

yme tested a

on of 4, 2,

din. Perform

d initial rat

gnal and su

vidin (Fig. S

manufacturer2 measured

complexes a

out 30 time

higher sign

version, sho

d with decr

nting a very

nzymes was

ml of biotiny

n trimmed

RP) or Hig

e and one

at each give

0.2 and 0.1

mance was t

te of substra

ubstrate con

S3). For the

r to bind fou

d the disso

and found th

s faster tha

nal obtained

owing the r

reasing conc

high turnov

tested. For

ylated detA

in short str

gh Sensitivi

control str

en concentr

1 μg/ml and

then compar

ate convers

nversion rat

tested, Avi

ur biotins pe

ociation rat

hat the disso

an that obse

with HSS-H

relevance o

centration o

ver number,

that purpos

Ab followin

rips and loa

ity Streptav

rip (withou

ation. HSN

d compared

red in respe

ion. High S

te compared

idin, Strepta

er molecule

te constant

ociation rat

erved for bi

HRP in a sh

S5

f washing

of antigens

, therefore

se, a given

ng similar

ading with

vidin–HRP

ut enzyme

-HRP and

d with the

ect to total

Sensitivity

d to High

avidin and

with high

ts of the

e constant

iotin from

hort period

 

Fig. S3

Sensitiv

ExtrAvi

Assay V

The assa

the lev

Absorba

samples

the met

%Accur

For each

values p

calculat

first valu

Th

of recom

was obt

the Intra

Quantifi

Compariso

vity NeutrAv

idin-HRP (E

Variability

ay sensitivit

vel above w

ance (Abs)

s. The Limit

thod descri

racy betwee

h experimen

plot was dra

ted. The LoQ

ue in which

he linear ran

mbinant pro

tained. The p

a-assay pre

fication, LLo

on of differe

vidin-HRP

EA-HRP). T

ty was asses

which sam

of the nega

t of Quantit

ibed by Ed

en the exper

nt, the expe

awn to find

Q was defin

h the Accura

nge was def

tein (IL-1β)

precision is

cision three

oQ, Middle

ent Peroxid

(HSN-HRP

The enzyme

ssed as LoD

mples were

ative contro

tation (LoQ)

derveen,3 w

rimental and

ected values

the linear r

ned as the co

acy was betw

fined as the

) for which

s herein refe

e samples w

e range, MR

de enzymes

P), High Sen

s were comp

D or “Cut-of

considered

ol plus three

) was also d

which uses

d expected

s were calcu

range. From

oncentration

ween 80-12

interval bet

an appropri

erred to as In

with differen

R; and near

tested for c

nsitivity Str

pared at sam

ff value” of

d positive,

e times the

determined

the best c

values calc

ulated and a

m this, the a

n correspon

0% and Pre

tween the lo

ate level of

ntra and Int

nt concentra

the Higher

colorimetric

reptavidin-H

me concentr

f the assay, w

and determ

standard d

for all the c

combination

culated using

an experime

accuracy and

nding to the

ecision <20%

ower and the

f precision, a

ter-assay pre

ations (near

Limit of Q

c amplificat

HRP (HSS-H

ration of 4 μ

which was d

mined as

eviation of

cytokines te

n of %Prec

g a calibrati

ental versus

d precision

Abs that rev

%.

e upper con

accuracy and

ecision. To

r the Lower

Quantificatio

S6

tion: High

HRP) and

μg/ml

defined by

the mean

the blank

sted using

cision and

ion curve.

s expected

were then

vealed the

ncentration

d linearity

determine

r Limit of

on, HLoQ,

S7  

all determined from the calibration curve) were tested in six replicates (6 strips with 10

capillaries; 60 capillaries in total). The average Abs values and the standard deviation for each

sample were calculated and the Coefficient of Variation (CV) was also determined for each

concentration within a given assay run. The Inter-Assay Calibration was determined by running

three assays in different days and using different MSA devices, in which samples were analysed

in duplicate, using the same three concentrations of recombinant proteins (corresponding to

LLoQ, MR and HLoQ). The average and the standard deviation for each sample were calculated

and the CV determined for each concentration between the assay runs. Typical CVs for ELISA

in 96-well MTP are in the range of 10–20%. 3,4

Accuracy or Recovery was also determined from the calibration curve by comparing the

expected value with the actual cytokine concentration in the assay. The expected versus the

average of the measured values was determined for each sample, by calculating the %Recovery=

assay value/expected value x 100. The typical range for accepted accuracies is 80-120%.3

Qualitative Duplex Assay

In order to demonstrate the ability of the new miniaturized platform to detect simultaneously

more than one cytokine a simple qualitative duplex assay was developed with of IL-1β and IL-6

reagents. For this purpose, solutions containing IL-1β or IL-6 capAb were injected into each

individual capillary using a small syringe needle. The MCF strip was then incubated for 2 hours

at room temperature, and then further incubated for 1.5 h with the blocking solution and washed

with PBS-T. The strips were then trimmed into 30 mm long individual test strips and attached

onto the MSA. Equal concentration of recombinant proteins (0.5 ng/ml) and detAb (10 g/ml)

were then used. All subsequent steps followed same sandwich ELISA procedure described in the

main manuscript.

Quantitative Triplex Assay

A quantitative triplex assay consisting of full response curve for each cytokine was performed

for simultaneous quantitation of IL-1β, IL-12 and TNFα. Individual capillaries on a 25 cm long

fluoropolymer MFC strip (containing 10 capillaries) were injected and incubated into one pair of

capillaries each with one of the following solutions: PBS (overall negative control), 3% BSA

(blocking solution control) or IL-1β, IL-12 capAb or TNFα at 20 µg/ml. All subsequent ELISA

S8  

steps were as already described for the singleplex and duplex assays, with the exception that

standard curves were prepared using a 1:3 dilution series of recombinant protein. All

recombinant proteins and detAb solutions for each cytokine were combined at same

concentration, which ultimately represents 3 times higher protein content on each solution well

when compared to singleplex detection. Combining different biotinylated detAb for multiplex

ELISA detection was found to significantly affect the individual cytokine performance by

increasing the background, therefore it was necessary to re-optimise the multiplex assay, in

respect to detAb and enzyme concentration, to maintain similar signal-to-noise ratios to

singleplex assays. This is described in Supplementary Results section.

SUPPLEMENTARY RESULTS

Assay Optimisation

The first two parameters tested were the incubation times of the capAb and recombinant

proteins. Two hours incubation at room temperature was sufficient to fully immobilize the capAb

by passive adsorption, as no differences were detected in signal strength and signal-to-noise ratio

(Fig. S4a). This had the advantage of saving the typical overnight incubation required for MTP

sandwich ELISAs. Equally, the incubation of recombinant protein for sensitive detection could

be reduced to 30 min in the fluoropolymer MCF without compromising sensitivity (Fig. S4b).

This is linked to the very short diffusion distances in the plastic microcapillaries. A parameter

found paramount in controlling the signal-to-noise ratio in the fluoropolymer MCF ELISA was

the blocking solution, for that reason few different formulations were tested. BSA and FBS are

commonly used for blocking non-specific binding sites in plastic surfaces. Although no

significant difference could be detected in respect to Abs signal intensity, the kinetic analysis of

the OPD conversion in the capillaries for different cytokine concentrations revealed poor

performance for both 1% BSA or 2.5% FBS in respect of background development. Fig. S4c

shows the initial rates of assay; in that plot the initial velocity v0 corresponded to the rate of

generation of absorbance in the MCF during the first few minutes of OPD conversion in the full

cytokine sandwich ELISA. BSA and FBS have similar proteins in size and molar rations on their

composition, since BSA is the main compound present in FBS; however on both cases a high

background was detected (Abs0≈0.05). In order to reduce the background which directly controls

S9  

to the sensitivity of the assays, a synthetic SuperBlock blocking solution from Thermo Scientific

was tested, which revealed lower backgrounds (Abs0≈0.02) (data not shown).

The use of higher capAb concentrations of 40 μg/ml and above resulted in increased

background and reduced signal (Fig. S4d), suggesting FEP antibody adsorption and/or

orientation was not favored by the presence of a very high capAb concentration. This is

presumably linked to the orientation of the surface adsorbed capAb molecules.5

The effect of detAb concentration was tested for a range between 0 and 20 μg/ml, and it

was also observed a benefit in using 10 μg/ml (Fig. S4e). Again, this is significantly higher than

the concentrations normally used for sensitive sandwich ELISA in MTPs and the increase in the

signal can be explained based on the same binding equilibrium principle. A large solution excess

of detAb favors the formation of the complex capAb-Ag-detAb at the surface of the plastic

capillaries, which is linked to the larger SAV ratio in small bore microcapillaries.

S10  

Fig. S4 Optimisation of IL-1 cytokine immunoassay measurement in the fluoropolymer MCF.

a) and b) show effect of of incubation time of capAb and recombinant protein, respectively. c)

Initial rates of colourimetric signal generation in the MCF strips for different blocking solutions

(BSA 1% and FBS 2.5%). d) Effect of capAb concentration (20 μg/ml). e) Effect of detAb

concentration for two different capAb concentration coatings. Assays conditions are detailed in

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.0 0.5 1.0

v0 (

ng

/ml.

s)

IL-1beta concentration (ng/ml)

BSA 1% TMB

2.5% FBS & 0.02%NaN3 TMB

C

0

0.1

0.2

0.3

0.4

0.5

0.6

0.0 2.5 5.0

Ab

s

IL-1beta concentration (ng/ml)

22.05.2013

23.05.2013

Overnight (fridge)

2 hours (RT)

A B

0.000

0.015

0.030

0.045

0.060

0 5 10 15 20 25

Ab

s

Detection Antibody concentration (ug/ml)

CapAb=10 ug/ml

CapAb=20 ug/ml

E

0

0.1

0.2

0.3

0.4

0.5

0.6

0.0 2.5 5.0

Ab

s

IL-1beta concentration (ng/ml)

1 hour

30 min

0.00

0.05

0.10

0.15

0.20

0.25

0 10 20 40 80 100

Ab

s

Capture Antibody concentration (μg/ml)

Exp Controls

D

S11  

Experimental Design section in the manuscript. The optimised concentrations for capAb and

detAb were considered 20 μg/ml and 10 μg/ml, respectively

Limit of Quantitation of the Colorimetric Detection Device

A series of dilutions of 2,3-diaminophenazine (DAP), the final product of the conversion of the

substrate OPD by the immunoassay enzyme HRP, were scanned in MCF with a flatbed scanner

starting at a concentration of 2 mg/ml, and in parallel peak absorbance (450 nm) of the same

dilutions was measured in a 96-well MTP using a microplate reader (Fig. S5). The DAP

absorbance in the blue channel of the scanned image was calculated by image analysis.

 

Fig. S5 Response curves for DAP detection in the MCF with flatbed scanner and 96 well MTP

with microplate reader (450 nm). Only concentrations corresponding to the range of DAP

concentration versus normalised Abs are presented for the MTP and fluoropolymer MCF

Qualitative Duplex Detection

To demonstrate the capability of simultaneous detection of two or more cytokines on each

fluoropolymer MCF strip, a duplex qualitative assay using IL-1β and IL-6 was performed and

analysed. All capillaries showed a positive color signal according to the capAb coating pattern

(Fig. S6), which confirms the possibility of detecting simultaneously more than one cytokine

from a single sample. The main assay conditions used on each MCF strip in the 8-channel MSA

device are shown in Fig. S6b. The higher signal observed for IL-1β cytokine was possibly due to

0.00024

0.0625

0.01

0.1

1

10

100

0.0001 0.001 0.01 0.1 1 10

Ab

s/c

m

DPA conc (mg/ml)

DAP MTP

DAP MCF

 

the fact

backgro

number

Fig. S6

and 4 w

with An

capillari

and 5 on

of IL-6

the sam

recombi

Supplem

Optimis

In order

of detA

that the opt

ound values

6 which sh

Qualitative

were coated

nti-Human I

ies 1, 9 and

nly 0.5 ng/m

recombinan

me concentra

inant prote

mentary Exp

sation of th

r to reduce t

Ab and Enzy

timisation w

s were in ge

owed increa

e duplex ass

with Anti-

IL-6 antibod

d 10 were o

ml of IL-1β

nt protein u

ation and th

ein). b) D

perimental D

he Triplex A

the backgrou

yme. A MC

was based on

eneral very

ased noise, a

say for dete

Human IL-

dy purified;

only blocked

recombinan

used. Strips

he strips 4 a

Duplex assa

Design secti

Assay

und, the trip

CF strip w

n this cytoki

y low with t

and the sign

ection of IL

-1β antibody

Capillaries

d for 1h30m

nt protein w

3 and 7 we

and 8 were

ay details.

ion

plex assay w

as coated w

ine, which l

the exceptio

nal-to-noise

L-1β and IL

y purified;

s 7 and 8 we

min (i.e. not

was added, a

ere filled w

used as con

The assa

was optimise

with 20 µg

led to IL-6 u

on of a sin

ratio (SNR

L-6 cytokine

Capillaries

ere incubate

t coated wit

and strips 2

ith both rec

ntrol strips

ay conditio

ed by testing

/ml of capA

underperform

ngle capillar

R) in the orde

es. a) Capill

5 and 6 we

ed only with

th capAb). I

and 6 only

combinant p

(i.e. with 0

ons are de

g different c

Ab of each

S12

ming. The

ry in strip

er of 5.

laries 2, 3

ere coated

h PBS and

In strips 1

0.5 ng/ml

proteins at

0 ng/ml of

etailed in

conditions

h cytokine

 

followin

Design

tested fo

Enzyme

cytokine

concent

detAb a

 

Fig. S7

assay co

HSS-HR

SUPPL

1 A2

2 U

3 J

4 J

ng the proce

section. Du

for the diffe

e, using 333

es tested).

trations givi

and 4 µg/ml

Comparison

onditions. a)

RP

LEMENTAR

A. I. Barbosa2928.

U. Piran and

. Ederveen,

. R. Crowth

edure descr

uplicated str

rent combin

3 pg/ml of

Several fu

ing the high

HSS-HRP

n of backgro

) 10 µg/ml d

RY REFER

a, A. P. Cas

d W. Riordan

A Practical

her, The ELI

ibed for qua

rips plus a

nations of 2

f recombina

ull response

her signal-to

(Fig. S7).

ound in Trip

detAb and 4

RENCES

stanheira, A

n, J. Immun

l Approach

ISA guidebo

antitative tr

blank strip

2.5, 5 and 1

ant protein

e curves w

o-noise ratio

plex Cytoki

4 µg/ml HSS

. D. Edward

nol. Methods

to Biologica

ook., 2000, v

riplex assay

(with no a

10 µg/ml of

(which fitte

were built

o. The optim

ine Assay us

S-HRP and

ds and N. M

s, 1990, 133

al Assay Va

vol. 149.

in Supplem

dded recom

f detAb and

ed the linea

using the

mised cond

sing optimis

b) 5 µg/ml d

M. Reis, Lab

3, 141–143.

alidation, Ho

mentary Exp

mbinant prot

d 1, 2 and 4

ar range of

detAb and

itions were

sed a) and in

detAb and 2

Chip, 2014

oofddorp, 2

S13

perimental

tein) were

4 µg/ml of

f all there

d Enzyme

10 µg/ml

 

nitial b)

2 µg/ml

4, 2918–

010.

S14  

5 M. E. Wiseman and C. W. Frank, Langmuir, 2012, 28, 1765–74.